PROJECT SUMMARY The proposed studies focus on the development of an efficacious, safe and easily administered malaria vaccine that can generate local and systemic protection against the pre-erythrocytic stages of Plasmodium. The feasibility of skin delivery of malaria vaccine is supported by the ability to elicit high levels of sterile immunity in human volunteers immunized by exposure to the bites of malaria-infected mosquitoes. The broader long-term goal is to use the well-defined circumsporozoite (CS) protein both as an immunogen and a model antigen to define the innate and adaptive immune responses elicited by skin scarification using a TLR agonist(s) adjuvant formulation as a proof-of-principle for the feasibility of transcutaneous malaria subunit vaccines. It is expected that the results of these studies will be directly applicable to all Plasmodium species, since the central repeat region of all Plasmodium CS proteins is a well defined target of potent sporozoite neutralizing antibodies. Moreover, these studies will support future development of combination vaccines containing multiple malaria antigens that are also targeted by high levels of humoral immunity, such as blood stages responsible for clinical disease and sexual stages that transmit the parasite to the mosquito vector to continue the parasite life cycle. In the proposed studies, we will combine dynamic and static microscopy, flow cytometry, and cytokine array analyses to define the spacio-temporal progression of the innate immune response that occur following skin scarification with malaria peptides and recombinant proteins (Aim 1), optimize humoral and cellular immunity using TLR agonists alone or in combination (Aim 1 & 2), and (Aim 3) explore the use of a large animal model, pigs, whose skin provides greater homology to human skin to advance translation towards human trials. More generally, mechanisms of skin scarification delivery and TLR agonist adjuvant formulations identified through these studies will also advance design of vaccines for other vector-borne diseases and skin-invasive parasites, as well as non-infectious diseases such as skin cancer.